Antibiotic b-2847rb and production thereof

ABSTRACT

A novel antibiotic B-2847RB, which is obtained by reducing antibiotic B-2847Y and/or antibiotic B-2847R, shows strong antimicrobial activities against Gram-positive bacteria and is characterized by a lessened toxicity against mammals and an increased stability in an aqueous solution in comparison with the antibiotics B-2847Y and B-2847R.

United States Patent [1 1 Kishi et al. 51 Feb. 20, 1973 [5 ANTIBIOTICB-2847RB AND [56] References Cited PRODUCTION THEREOF UNITED STATESPATENTS [75] Inventors: Toyokazu Kishi, Nara; Mitsuko Asai, Takatsuki;Setsuo Harada; 3,014,841 et al. Muroi of Komei Mizuno, Settsu, all ofJapan Pri E er-Al tT. rs [7 3] Assignee: Takeda Chemical Industries,Ltd., A s 22 3 23 gigg Osaka Japan AttorneyWenderoth, Lind & Ponack [22]Filed: March 31, 1970 21 Appl. No.: 24,130 [57] ABSTRACT A novelantibiotic B-2847RB, which is obtained by [30] Foreign ApplicationPriority Data reducing antibiotic B-28 47 Y and/or antibiotic B- 2847R,shows strong antimicrobial activities against March 31, JapanGram-positive bacteria and is characterized a lessened toxicity againstmammals and an increased sta- US. Cl. in an aqueous olution incomparison the [51] Int. Cl. ..A6lk 21/00 antibiotics 47 and 2347R [58]Field of Search ..424/122-124 2 Claims, 2 Drawing FiguresPATENTEDFEBZO'W 5.717. 707

SHEET 10F 2 7(M ILLIMICRONS) F 1G .I

INVENTORS TOYOKAZU KISHI TSUKO ASA! SETSUO HARADA MASAYUKI MUROI K6ME|MIZUNO BY z/wld, $4 1M ATTORNEYS ANTIBIOTIC B-2847RB AND PRODUCTIONTHEREOF This invention relates to antimicrobially active novel compoundand to production thereof. More particu- 5 larly, this invention relatesto antibiotic B-2847RB and production thereof.

The present inventors have found that when antibiotics B-2847Y andB-2847R, both being discovered by the present inventors together withsome other researchers (See Dutch Patent application No. 68,02679 laidopen into public inspection on Aug. 26, 1968), are subjected toreduction with a specific reducing agent, a novel antibiotic showing alessened toxicity and an increased stability is obtained. This novelantibiotic has been named B-2847RB in contrast to the known antibioticsB-2847Y and B-2847R.

The starting materials of the present invention, B-

2847Y and B-2847R have also been disclosed in U.S.

application Ser. No. 708,319 filed Feb. 26, 1968, now U.S. Pat. No.3,627,881, issued Dec. 14, 1971, which Patent is commonly owned by theAssignees of the present invention. Further, the context of the U.S. ap-

plication Ser. No. 708,319 is essentially the same as the Dutchapplication No. 6,802,679 mentioned above and describes in detail how toprepare the B-2847Y and B- 2947R materials.

The principal object of the present invention is to provide a novelantibiotic B-2847RB with excellent properties. Another object of thepresent invention is to provide an industrially feasible method forproducing the said antibiotic B-2847RB.

Antibiotics B-2847Y and B-2847R to be employed as the starting materialsin the method of the present invention may be prepared by, for example,cultivating Streptomyces tolypophorus ATCC-21 177 as disclosed in thesaid Dutch Patent application No. 68,02679.

in the method of the present invention, antibiotic B- 2847Y, antibioticB-2874R or a mixture thereof is subjected to reduction to yieldantibiotic B-2847RB. As the reducing agent, there may be advantageouslyemployed a borohydride e.g. sodium borohydride, or a dithionite e.g.sodium dithionite. This reduction reaction smoothly proceeds at a roomtemperature (about 5 to 30 C). As the solvent, there may preferably beemployed one or more organic solvents, in which the starting materialscan be dissolved, or a mixture of water and such an organic solvent. 5

C H 6l.42i-l.0% 6.8510576 2. Specific Rotation 3. Color Reaction It ispositive to ferric chloride-potassium ferricyanide reagent (1:1 byweight) 4. Molecular Weight About 830 when measured by the VaporPressure method using ethyl acetate as a solvent. 5. Solubility Solublein methanol, ethanol, n-butanol, acetone, chloroform, ethyl acetate,diethyl ether; hardly soluble in water, and insoluble in n-hexane. 6.Absorption Spectrum The ultraviolet and visible absorption spectrum inethanol is as shown in FIG. 1 of the accompanying drawing.

The significant maximum absorptions observed are as follows:

240i2mp. (E, 426:40)

30li2mp. E,,,,, 197x20 Infrared absorption spectrum in chloroform is asshown in FIG. 2 of the accompanying drawing, and the significantabsorptions in wave number are as follows: 3490(M), 3000(M), 1715(M),1670(8), 1625(8), 1565(Sh), 1530(VS), 1465(V8), 1385(8), 1370(8),1325(8), 1280(8), 1240(V8), 1160(8), 1095(8), 1065(8), 1020(8h),1003(M), 973(8), 945(M), 920(M), 898(M), 885(8h)cm.

Notes: VS; very strong, 8; strong, M; medium, 8h;

shoulder.

The biological properties of the antibiotic B-2847RB are as follows:

1. Antimicrobial spectrum Antimicrobial activities of B-2847RB againstvarious microorganisms are shown in Table 1.

The tests are carried out at 37 C on bouillon agar for 20 hours againstordinary bacteria, at 37 C on glycerin bouillon agar for 48 hoursagainst acid-fast bacteria.

As clearly seen in Table l, antibiotic B-2847RB shows strongantibacterial activities against Gram-positive bacteria.

2. Acute toxicity Fifty percent Lethal doses (LD of the antibiotic B-2847RB in mice are as follows;

1000 mg/kg. (intraperitoneally, when observed for 7 days).

about 500-750mg/kg. (intraveneously, when observed for 7 days).

The novel Antibiotic B-2847RB has clearly different properties fromthoseof Antibiotics B-2847Y and R, among others, in ultraviolet spectra,infrared spectra and specific rotation.

Therapeutic tests involving the oral administration of testantibioticsto infected animals have revealed that B-2847RB is substantially aseffective as B-2847Y and B-2847R.

Furthermore, antibiotic B-2847RB is characterized by a lessened toxicityagainst mammals and an increased stability in an aqueous solution incomparison with B-2847Y and B-2847R.

Staphylococci which are pyogenic or pus-forming tend to producecircumscribed lesions, e.g. in the form of abscesses and the like, whichoften occur in the skin. Those Staphylococci are the cause of furunclesand of .carbuncles and other common wound infections. An-

tibiotic B-2847RB of the present invention is useful in pharmaceuticalpreparations for the topical treatment of this type of infection inmammals (dogs, cats, humans, etc.). Thus, a useful preparation fortopical application to an infection due to Staphylococcus aureus isasfollows:

Into 1 gram of wool fat is uniformly incorporated 10 to 20 milligrams ofantibiotic B-2847RB and the mixture is then admixed uniformly withsufficient white petrolatum to make 10 grams of an ointment.

This ointment is to be applied topically in an amount sufficient tocover the wound or region to be treated, with gently rubbing in, atleast once daily or, depending on cases, severaltimes daily.

Due to the above-mentioned bacteriocidal activity of antibioticB-2847RB, it is useful e.g. to disinfect hospital apparatus, etc. whichare generally exposed to pathogenic Gram-positive bacteria of the typewhich are sensitive to this product, as aforementioned. Disinfection iseffected by application or spraying of a solution (e.g. methanolic orethanolic, etc.) containing about 20 to 200 micrograms of antibioticB-2847RB.

The following examples are merely for illustrative purposes and are notto be construed as limitation of the present invention. In theseexamples, the abbreviations mg., g., ml. refer respectively tomilligram(s), gram(s) milliliter(s) and parts by weight bear the samerelation to parts by volume as do grams to milliliters.

EXAMPLE 1 under reduced pressure. The resulting residue is dis solved in30 ml. of acetone. The solution is admixed with 150 ml. of n-hexane togive precipitates. The precipitates are recovered by filtration to yield3.0g. of crude powder.

A solution of 1.0g. of the crude powder in 10 ml. of ethyl acetate isallowed to pass through a column packed with 20g. of silica gel and thecolumn was subjected to elution with 400 ml. of a mixture of ethylacetate and acetone (4:1 by volume). The eluate is concentrated todryness and the residue is dissolved in 5 ml. of benzene. To theresulting solution is added 50 ml. of n-hexane to give 0.8g. of yellowpowder of antibiotic B-2847RB.

0.6g. of the powder is subjected to thin layer chromatography on silicagel employing a mixture of ethyl acetate and acetone (1:1 by volume) asthe developer. The yellow zone at Rf about 0.65 is extracted with ml. ofethyl acetate. The extract is concentrated, followed by the addition ofn-hexane to give 0.4g. of antibiotic B-2847RB as yellow powder.

EXAMPLE 2 To a solution of 300 mg. of antibiotic B-2847Y in 30 ml. ofacetone is added at a room temperature little by little 10 ml. of a 10percent aqueous solution of sodium dithionite (biz 1 8 0 211 0) and themixture is kept standing with stirring for 15 minutes. The reactionmixture is subjected to extraction with 30 ml. of benzene. The benzenelayer is washed with water and is concentrated under reduced pressure.

The resulting residue is subjected to thin layer chromatography onsilica gel containing 2 percent of oxalic acid employing a mixture of 1percent oxalic acid-containing ethyl acetate and acetone (1:1 vbyvolume) as the developer. The yellow zone at Rf about 0.5 is extractedwith 100 ml. of ethyl acetate. The extract is concentrated to about 1ml. To the concentrate is added 20 ml. of n-hexane to give 30 mg. ofantibiotic B- 2847RB as yellow powder.

EXAMPLE 3 To a solution of 500 parts by weight of antibiotic B- 2847R in100,000 parts by weight of ethyl acetate is added 40,000 parts by volumeof water. With the mixture being stirred at a room temperature, 250parts by weight of sodium borohydride is added thereto little by little.After stirring for 20 minutes, the ethyl acetate layer is recovered andneutralized with ZN-hydrochoric acid and subsequently washed with water.After dehydration, the ethyl acetate layer is concentrated under reducedpressure. The resulting residue is dissolved in 2000 parts by volume ofbenzene. The solution is admixed with 20,000 parts by volume of n-hexaneto give 340 parts by weight of anti-biotic B- 2847RB.

What is claimed is:

l. Antibiotic B-2847RB, having the following characteristics:

1. Elementary analysis is C 61.42" *-l.0%, H

2. Specific rotation is [011 +133.4:':13(C

3. Color reaction is positive to ferric chloride-potassium ferricyanidereagent (1:1 by weight);

4. Molecular weight is about 830 by the Vapor Pressure method;

5. Soluble in methanol, ethanol, n-butanol, acetone,

chloroform, ethyl acetate, diethyl ether, benzene; hardly soluble inwater; and insoluble in n-hexane;

6. Ultraviolet and visible absorption spectrum in EtOH is as shown inFIG. 1 of the accompanying drawing, and the significant maximumabsorptions observed are as follows:

240mm E,,,,, 426:40)

301i2mp. E,,,,, 197:20

7. Infrared absorption spectrum is as shown in FIG. 2 of theaccompanying drawing, with the following significant absorption in wavenumber:

3490(M), 3000(M), 1715(M), 1670(5), 1625(8), 1565(Sh), 1530(VS),1465(VS), 1385(8), 1370(8),

1325(8), 1280(8), 1240(VS), 1160(8), 1095(8), 1065(S), 1020(Sh),1003(M), 973(8), 945(M),

1. Antibiotic B-2847RB, having the following characteristics: 1.Elementary analysis is C 61.42 + or - 1.0%, H 6.85 + or -0.5%, N 3.31 +or - 0.5%;
 3. Color reaction is positive to ferric chloride-potassiumferricyanide reagent (1:1 by weight);
 4. Molecular weight is about 830by the Vapor Pressure method;
 5. Soluble in methanol, ethanol,n-butanol, acetone, chloroform, ethyl acetate, diethyl ether, benzene;hardly soluble in water; and insoluble in n-hexane;
 6. Ultraviolet andvisible absorption spectrum in EtOH is as shown in FIG. 1 of theaccompanying drawing, and the significant maximum absorptions observedare as follows: lambda max 224 + or - 2m Mu (Elcm1% 446 + or - 45) 240 +or - 2m Mu (Elcm1% 426 + or - 40) 301 + or - 2m Mu (Elcm1% 197 + or -20) 420 + or - 2m Mu (Elcm1% 203 + or - 20)
 7. Infrared absorptionspectrum is as shown in FIG. 2 of the accompanying drawing, with thefollowing significant absorption in wave number: 3490(M), 3000(M),1715(M), 1670(S), 1625(S), 1565(Sh), 1530(VS), 1465(VS), 1385(S),1370(S), 1325(S), 1280(S), 1240(VS), 1160(S), 1095(S), 1065(S),1020(Sh), 1003(M), 973(S), 945(M), 920(M), 898(M), 885(Sh)cm 1 (inCHCl3); and
 8. Shows antimicrobial activities against Gram-positivebacteria.